High glucose-induced IKK-Hsp-90 interaction contributes to endothelial dysfunction.
A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor kappaB kinases (IKK)alpha, beta, and gamma] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKbeta in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKbeta interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 +/- 0.65-fold) and translational (1.97 +/- 0.17-fold) expression as well as the catalytic activity of IKKbeta (2.45 +/- 0.4-fold). Both IKKbeta and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 microM) or Radicicol (20 microM) mitigated (0.45 +/- 0.04-fold and 0.93 +/- 0.16-fold, respectively) HG induced-IKKbeta activity (2.5 +/- 0.42-fold). Blocking of IKKbeta expression by IKK inhibitor II (15 microM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.
Mohan
Sumathy
S
Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229, USA. mohan@uthscsa.edu
Konopinski
Ryszard
R
Yan
Bo
B
Centonze
Victoria E
VE
Natarajan
Mohan
M
D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology; D004790:Enzyme Induction; D031541:Fluorescence Resonance Energy Transfer; D005947:Glucose / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006943:Hyperglycemia / Q000378:metabolism* / Q000503:physiopathology; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D020798:Two-Hybrid System Techniques
D016227:Benzoquinones; D003374:Coumarins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018942:Macrolides; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C051122:wedelolactone; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051550:I-kappa B Kinase; C035359:monorden; D005947:Glucose; C001277:geldanamycin